Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng J. et al., J. Biol. Chem., 1992, 267, 7402-05). Exendin-4 is an activator of the glucagon-like peptide-1 (GLP-1) receptor, whereas it shows only very low activation of the GIP receptor and does not activate the glucagon receptor (see Table 1).
TABLE 1Potencies of exendin-4 at human GLP-1, GIP and Glucagon receptors (indicated in pM) at increasing concentrations and measuring the formed cAMP as described in Methods.SEQ IDEC50 hGLP-1 REC50 hGIP R EC50 hGlucagon RNO:peptide[pM][pM][pM]1exendin-40.412500.0>10000000
Exendin-4 shares many of the glucoregulatory actions observed with GLP-1. Clinical and non-clinical studies have shown that exendin-4 has several beneficial antidiabetic properties including a glucose dependent enhancement in insulin synthesis and secretion, glucose dependent suppression of glucagon secretion, slowing down gastric emptying, reduction of food intake and body weight, and an increase in beta-cell mass and markers of beta cell function (Gentilella R et al., Diabetes Obes Metab., 11:544-56, 2009; Norris S L et al., Diabet Med., 26:837-46, 2009; Bunck M C et al., Diabetes Care., 34:2041-7, 2011).
These effects are beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of getting diabetes, hypertension, hyperlipidemia, cardiovascular and musculoskeletal diseases.
Relative to GLP-1 and GIP, exendin-4 is more resistant to cleavage by dipeptidyl peptidase-4 (DPP4) resulting in a longer half-life and duration of action in vivo (Eng J., Diabetes, 45 (Suppl 2):152A (abstract 554), 1996; Deacon C F, Horm Metab Res, 36: 761-5, 2004).
Exendin-4 was also shown to be much more stable towards degradation by neutral endopeptidase (NEP), when compared to GLP-1, glucagon or oxyntomodulin (Druce M R et al., Endocrinology, 150(4), 1712-1721, 2009).
Nevertheless, exendin-4 is chemically labile due to methionine oxdiation in position 14 (Hargrove D M et al., Regul. Pept., 141: 113-9, 2007) as well as deamidation and isomerization of asparagine in position 28 (WO 2004/035623).
The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 2:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
Liraglutide is a marketed chemically modified GLP-1 analogue in which, among other modifications, a fatty acid is linked to a lysine in position 20 leading to a prolonged duration of action (Drucker D J et al, Nature Drug Disc. Rev. 9, 267-268, 2010; Buse, J B et al., Lancet, 374:39-47, 2009).
The amino acid sequence of Liraglutide is shown as SEQ ID NO: 3:
HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoylamino-butyryl-)EFIAWLVRGRG-OH
Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown as SEQ ID NO: 5:
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH
During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Recent publications suggest that glucagon has in addition beneficial effects on reduction of body fat mass, reduction of food intake, and increase of energy expenditure (K M Heppner, Physiology & Behavior 2010, 100, 545-548).
GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acid peptide that is released from intestinal K-cells following food intake. GIP and GLP-1 are the two gut enteroendocrine cell-derived hormones accounting for the incretin effect, which accounts for over 70% of the insulin response to an oral glucose challenge (Baggio L L, Drucker D J. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; 132: 2131-2157).
GIP's amino acid sequence is shown as SEQ ID NO: 4:
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH
Holst (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728) describe that GLP-1 receptor agonists, such as GLP-1, liraglutide and exendin-4, improve glycemic control in patients with T2DM by reducing fasting and postprandial glucose (FPG and PPG). Peptides which bind and activate the GLP-1 receptor are described in patent applications WO 98/08871 A1, WO2008/081418 A1 and WO2008/023050 A1, the contents of which are herein incorporated by reference.
Designing hybrid molecules which combine agonism on the GLP-1 receptor, the glucagon receptor and optionally the GIP receptor offers the therapeutic potential to achieve better reduction of blood glucose levels, increased insulin secretion and an even more pronounced significant effect on body weight reduction compared to the marketed GLP-1 agonist Liraglutide (Hjort et al. Journal of Biological Chemistry, 269, 30121-30124, 1994; Day J W et al, Nature Chem Biol, 5: 749-757, 2009).
Peptides which bind and activate both the GLP-1 and the glucagon receptor and optionally the GIP receptor, and improve glycaemic control, suppress body weight gain and reduce food intake are described in patent applications WO 2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO 2011/075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO 2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/087671, WO 2011/087672, WO2011/117415, WO2011/117416, WO 2012/177443 WO 2012/177444, WO 2012/150503, WO 2013/004983, WO 2013/092703, WO 2014/041195 and WO 2014/041375, the contents of which are herein incorporated by reference. These applications disclose that mixed agonists of the GLP-1 receptor, the glucagon receptor and optionally the GIP receptor can be designed as analogues of the native GLP-1 or glucagon sequences.
Bloom et al. (WO 2006/134340) disclose that peptides which bind and activate both the glucagon and the GLP-1 receptor can be constructed as hybrid molecules from glucagon and exendin-4, where the N-terminal part (e.g. residues 1-14 or 1-24) originates from glucagon and the C-terminal part (e.g. residues 15-39 or 25-39) originates from exendin-4. Such peptides comprise glucagon's amino acid motif YSKY in position 10-13. Krstenansky et al (Biochemistry, 25, 3833-3839, 1986) show the importance of these residues 10-13 of glucagon for its receptor interactions and activation of adenylate cyclase.
In the exendin-4 derivatives described in this invention, several of the underlying residues are different from glucagon and the peptides described in WO 2006/134340. In particular residues Tyr10 and Tyr13, which are known to contribute to the fibrillation of glucagon (D E Otzen, Biochemistry, 45, 14503-14512, 2006) are replaced by Leu in position 10 and Gln, a non-aromatic polar amino acid, in position 13. This replacement, especially in combination with isoleucine in position 23 and glutamate in position 24, leads to exendin-4 derivatives with potentially improved biophysical properties as solubility or aggregation behaviour in solution. The non-conservative replacement of an aromatic amino acid with a polar amino acid in position 13 of an exendin-4 analogue surprisingly leads to peptides with high activity on the glucagon receptor, keeping their activity on the GLP-1 receptor (see also WO2013/186240.
Compounds of this invention are exendin-4 derivatives, which not only show agonistic activity at the GLP-1 receptor but also at the glucagon receptor and optionally the GIP receptor and which have only 4 or 5 amino acid exchanges compared to native exendin-4 including—amongst others—in position 14 an amino acid substituted with a lipophilic side-chain (e.g. a fatty acid combined with a linker).
Surprisingly, it was found that the modification of the amino acids in position 2 and 3 of native exendin-4 in combination with a fatty acid acylated residue in position 14 leads to peptides with a significantly higher glucagon receptor activity than the corresponding peptides with otherwise identical amino acid sequence with methionine (as in exendin-4) or leucine in position 14 (see Table 7). Additionally, this fatty acid functionalization in position 14 results in an improved pharmacokinetic profile.
Compounds of this invention are more resistant to cleavage by neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP4), resulting in a longer half-life and duration of action in vivo, when compared with native GLP-1 and glucagon.
Compounds of this invention preferably are soluble not only at neutral pH, but also at pH 4.5. This property potentially allows co-formulation for a combination therapy with an insulin or insulin derivative and preferably with a basal insulin like insulin glargine/Lantus®.